Science China Life Sciences

Although Cinnamomum includes globally important spice and medicinal plants, genome-scale knowledge of Cinnamomum species native to Japan remains limited. The Nansei Islands are an approximately 1,200-km-long archipelago located between the Pacific Ocean and the East China Sea and represent a key biogeographic region shaped by deep-sea barriers such as the Tokara and Kerama Gaps. Here, we integrated chloroplast and nuclear genome data generated in this study with public datasets to examine the phylogenetic relationships of four Cinnamomum species native to the Nansei Islands and ten related species. Chloroplast genomes showed no major structural differences among the four island species, whereas nuclear genome data resolved two distinct evolutionary lineages in the Nansei Islands: C. yabunikkei, C. x durifruticeticola, and C. daphnoides formed a distinct Nansei Islands lineage separate from Sri Lankan, Indonesian, and Taiwanese lineages, whereas C. sieboldii was placed in a Taiwanese lineage. Ancestry estimation indicated that C. x durifruticeticola and C. daphnoides share a common ancestral component, while C. sieboldii exhibited a single homogeneous ancestral component, suggesting reduced ancestral diversity after divergence from a Taiwanese lineage. These findings reveal previously unrecognized genomic differentiation among Japanese Cinnamomum species and suggest that deep-sea barriers, dispersal, and long-term isolation shaped their diversification.

Porcine deltacoronavirus (PDCoV) is a globally circulating coronavirus of swine that causes severe enteric disease and mortality in neonatal piglets. Its broad host range and zoonotic potential raises concerns about continued spillover into human populations. However, the host factors that govern PDCoV infection remain largely unexplored. Identifying host factors involved in virus infection may uncover novel therapeutic targets for antiviral drug development or targets for gene editing to create resistant pigs. Here, we conducted a genome-wide CRISPR knockout (GeCKO) screen to identify host factors contributing to PDCoV infection in swine testicular (ST) cells. Small guide RNAs (sgRNAs) targeting the ANPEP gene, expressing the main receptor porcine aminopeptidase N for PDCoV, were enriched in PDCoV-resistant cells, demonstrating the validity of the screen. Moreover, we identified transmembrane protein 30A (TMEM30A), an accessory {beta}-subunit of the P4-ATPase flippase complex, as a host dependency factor for PDCoV infection along with several potential PDCoV-induced cell death regulators, including DET1, IFNAR1, RFWD2, STAT1 and TP53. We further demonstrated that PDCoV attachment and internalization into cells were significantly impaired in TMEM30A knockout ST cells, suggesting that TMEM30A is important in the early stages of the PDCoV life cycle. Because TMEM30A also contributes to SARS-CoV-2 replication, future studies are warranted to investigate its mechanistic role in the context of coronavirus infection.

A central question in developmental biology is how spatiotemporal embryo morphological events and cell differentiation are precisely coordinated to eventually develop into a mature organism. Previous research has shown cell positions and asymmetric division stages are related to cell lineage specification since morula stage. However, the detailed spatiotemporal dynamics and histories of embryonic cells and their relations with cell lineage specification remain incompletely understood from the onset of embryo development onward. My study on in-vitro embryos showed that continuous live cell tracking mapped community-like patterns in cell origin and differentiation. Cell positions, cell temporal factors (such as cell division order and cell cycle) and their histories intricately interacted over pre- and peri-implantation stages. These interactions of spatiotemporal cellular activities directed embryo morphological events and asymmetric cell lineage origin and differentiation throughout 2-cell to around 100-cell stage. In conclusion, this study provides comprehensive insights into the cellular spatiotemporal dynamics of cell inheritance and differentiation during pre- and peri-implantation morphological events and cell lineage specification. By longitudinally integrating spatial and temporal parameters relating to early embryogenesis, the findings not only bridge divergent explanations stemmed from different studies in the field but also offers a refined foundation for evaluating embryo potential and improving outcomes in assisted reproductive technologies, stem cell research, and regenerative medicine.

The long-range RNA-RNA interaction between the termini of a flavivirus genome is controlled by multiple local RNA secondary and tertiary structures. However, a complete picture of this essential genome cyclization process is still elusive. Herein, by using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), SHAPE and mutational profiling, in vitro RNA binding assays, and reverse genetics approaches, we demonstrated that the hairpin 2 in the capsid-coding region (cHP2) is required for efficient replication of yellow fever virus and functionally interacts with the 3'-small hairpin (sHP) during genome cyclization. Interestingly, through a combination of multidimensional chemical probing and functional characterization assays, it was discovered that the sHP forms previously unknown tertiary interactions with an immediately upstream GACG region embedded within the 3'-cyclization sequence. This crucial tertiary RNA motif accounts for a potential negative regulatory mechanism of genome cyclization and is universally conserved among the mosquito-borne-related flaviviruses, which constitute a major phylogenetic branch within the subgenus Euflavivirus, genus Orthoflavivirus. Our study highlights the intricacy of RNA-based regulation in flaviviruses.

Epstein-Barr virus (EBV) infects 90% of the population worldwide and is associated with multiple malignancies, including Burkitt, Hodgkin and post-transplant lymphomas, nasopharyngeal and gastric carcinoma. EBV also triggers autoimmune diseases, including multiple sclerosis and systemic lupus erythematosus. While host factors important for EBV B-cell entry have been defined, including CD21/CR2, MHC class II and R9AP, much remains to be learned about host factors that support post-entry steps of EBV infection. To gain insight, we conducted a human genome-wide CRISPR-Cas9 screen. In addition to known EBV B cell receptors/co-receptors, we identified multiple new factors and pathways essential for EBV B cell infection. These included multiple B cell signaling pathways, actin cytoskeleton and nuclear import factors. Knockout of CD19, its tetraspanin chaperone CD81 or karyopherin subunit alpha 1 (KPNA1, also called importin-5) significantly impaired establishment of EBV infection. EBV and CD19 co-localized at early stages of EBV infection, and the CD19 cytoplasmic tail was important for EBV infection, suggesting CD19 signaling supports EBV uptake. Depletion of KPNA1 significantly impaired establishment of EBV infection, but did not impair production of infectious EBV upon reactivation of latently infected B cells. Together, these results provide insights into pathways that support EBV B cell infection and identify potential therapeutic targets.

The resurgence of the Mpox virus (MPXV) highlights the urgent need for scalable, mutation-resistant countermeasures. Slow development timelines hinder traditional empirical vaccine discovery, while conventional vaccinology often yields high false-positive rates because it relies on static, linear sequence-based screening. These classical informatics filters consistently fail to predict physical structural stability or the dynamic presentation of immune cells under physiological conditions, creating a distinct translational gap between computational design and in vitro efficacy. To address these limitations, here we engineered an advanced, AI-augmented vaccine discovery pipeline targeting the most virulent components of the MPXV proteome. We systematically screened eight critical viral targets essential for replication, membrane morphogenesis, and host-cell entry to map highly conserved T-cell and B-cell epitopes. Moving beyond standard heuristic filtering, we implemented a high-fidelity deep learning validation layer: ESM-2 protein language models (pLMs) were deployed to calculate pseudo-perplexity scores across chimeric linker junctions (EAAAK, GPGPG) to guarantee structural naturalness, MHCflurry ensemble models successfully verified active intracellular antigen presentation, and structure-informed graph neural networks (GNNs) simulated the physical node-attention dynamics of the immune synapse. Finally, recurrent neural networks (RNNs)-driven mRNA kinetic modeling optimized translation initiation rates and minimum free energy profiles during in silico cloning. This multi-layered pipeline successfully yielded three structurally refined, multi-epitope vaccine constructs optimized for maximum global population coverage. ESM-2 structural validation confirmed exceptionally low perplexity scores at structural junctions, indicating that the engineered chimaeras have native-like stability and a minimized risk of proteolysis. GNN-based immune synapse modeling demonstrated highly stable binding kinetics and robust binding free energies between the target epitopes and human HLA alleles. Furthermore, RNN kinetic optimizations established that the cDNA sequences are optimized for high-yield expression in heterologous expression hosts without risk of ribosomal stalling or translational bottlenecks. This study establishes a deep learning-augmented framework that bridges the gap between theoretical sequence optimization and biopharmaceutical scalability. By validating the structural naturalness, real-world immunogenicity, and manufacturing kinetics of our vaccine chimeras, this framework presents a robust, proactive paradigm for rapid vaccine architecture against emerging orthopoxvirus threats.

Oysters are vital to global aquaculture and marine ecosystems, yet, their associated RNA viruses remain largely unexplored. Here, we analysed ~1,000 publicly available metatranscriptomic datasets from 13 oyster species across 14 countries and 5 continents to characterize the oyster RNA virome (ORV). We identified 1,515 distinct RNA viruses putatively representing 17 viral orders and 766 species-level viral operational taxonomic units (vOTUs), with ~62% being previously undocumented putative species. These viruses not only populate extant branches of RNA virus evolution, but also represent previously unrecognized families and genera, and include viruses that putatively infect bacteria, fungi, protists, plants, invertebrates, and vertebrates. Based on sequence similarity, we found many calicivirus-like, orthoreovirus-like, astrovirus-like, and picornavirus-like viruses that do not infect invertebrates or microbes; thus, oysters accumulate diverse viruses from the surrounding water, and can potentially be interrogated as reporters of viruses circulating in the environment.

Protein kinase CK2 (CK2), a ubiquitously expressed serine/threonine kinase, has emerged as a key host factor exploited by several viruses, including Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we investigate the role of CK2 subunits in SARS-CoV-2 replication and evaluate the antiviral potential and broad-spectrum anti-coronavirus activity of two CK2 inhibitors: the previously described ATP-competitive inhibitor CX-4945 and a newly developed, highly selective bivalent inhibitor, AB668. The siRNA-mediated knockdown of CK2 subunits significantly reduced SARS-CoV-2 replication, highlighting its essential role in the viral lifecycle. Co-immunoprecipitation assays revealed that the SARS-CoV-2 nucleoprotein interacts with CK2. Furthermore, bio-layer interferometry assays demonstrated that the nucleoprotein specifically binds to the tetrameric CK2 holoenzyme (CK22{beta}2) but not to individual CK2 or CK2{beta} subunits. Pharmacological inhibition of CK2 with AB668 exhibited potent antiviral activity, significantly reducing viral replication by targeting a pre-replicative or RNA-replicative step of the viral life cycle. Importantly, AB668 displayed broad-spectrum antiviral activity, effectively inhibiting both HCoV-OC43 ({beta}-CoV) and HCoV-229E (-CoV). In summary, our findings establish CK2 as a critical host factor for SARS-CoV-2 replication and identify AB668 as a promising candidate for broad-spectrum antiviral therapy. With its high selectivity, minimized off-target effects, and efficacy against multiple coronaviruses, AB668 represents a valuable chemobiological and therapeutic tool.

Genetically modified immune effector cells, including chimeric antigen receptor (CAR) T cells, have emerged as transformative anti-cancer agents. However, these autonomous, living therapies are limited by difficult-to-control hyperactivation toxicities. To achieve their full potential as therapies for a broader range of diseases, CAR T cells must target novel antigens and integrate further potency enhancements that raise the possibility of novel and intensified toxicities. High performance safety systems are needed to restrain these highly active next-generation gene- and cell-based therapies. Cell therapy suicide switches remain infrequently used, and are currently limited by inaccessible controller drugs, incomplete cell depletion, fitness cost, and immunogenicity. To address these issues, we engineered a fully human sequence-based cell therapy suicide switch based on the overexpression of Caspase-Activated DNase (CAD) and its stoichiometric chaperone/inhibitor ICAD fused to a lenalidomide-inducible degron. Acute destabilization of ICAD with the broadly available, off-patent molecular glue degrader drug lenalidomide uncaged CAD to induce DNA damage and cell death. By this degradable cage mechanism, the ICAD-CAD suicide switch achieved long-term expression in primary human T cells without detectable leakiness, and deep drug-induced cell depletion. The ICAD-CAD switch was generalizable to alternative acute destabilization systems. Genome-wide CRISPR screening established the necessity of CAD and the lenalidomide-interacting E3 ubiquitin ligase substrate adaptor CRBN for switch function. When co-delivered with a CAR, ICAD-CAD CAR T cells retained effector functions and could be rapidly depleted with lenalidomide.

The 2026 Andes virus (ANDV) outbreak aboard the MV Hondius cruise ship resulted in 11 confirmed cases and 3 deaths and raised questions regarding the origin of infection, with much media focus resting on a landfill in Tierra del Fuego. We evaluated competing exposure hypotheses by assembling open data on reservoir host occurrences, rodent serosurveys, and historical ANDV outbreaks along with the index case's self-documented, publicly-posted citizen-science digital record of their movements. Historical outbreaks associated with human-to-human transmission were concentrated within a restricted region spanning southern Chile and adjacent western Argentina, coinciding with the distribution of the principal reservoir host, Oligoryzomys longicaudatus, and areas identified as elevated risk by multiple ecological and genetic models. The index case was documented within this transmission focus shortly before the putative exposure period. Together, our open data approach was able to rapidly locate initial exposure within the established ANDV transmission focus and develop scenarios to prioritise further epidemiological investigation.

Synthetic allopolyploidization is a powerful strategy for generating strains with novel industrially relevant traits. During the design of select, hybridize, evolved and learn cycle, the mitochondrial inheritance is considered neutral and most studies overcome their phenotypic impact. Here, we generated 168 synthetic allotetraploid strains representing all pairwise parental combinations of eight wild Saccharomyces species, and systematically controlled mitochondrial inheritance in these crosses. These allopolyploids were phenotyped across 31 environmental conditions relevant to biotechnology. In ~50% of all condition-strain combinations conditions, allotetraploids exhibited mid-parent heterosis. In approximately 25% of conditions, we found crosses with hybrid vigor, particularly in combinations involving Saccharomyces arboricola x Saccharomyces mikatae . However, allotetraploids were highly unstable and rapidly sporulated. Our analyses revealed that environmental conditions accounted for the largest effects, follow by its interaction with mitotype. However, mitotype alone showed small effects but significantly affected 42% of the tested conditions, underscoring the importance of mitochondrial inheritance as a key determinant of allopolyploid performance. These findings demonstrate that mitochondrial inheritance is a key, environment-dependent determinant of allopolyploid fitness and should be considered alongside nuclear genome composition in the rational design of industrial allopolyploid strains

Cell-surface glycoRNAs participate in diverse biological processes, but they were investigated as pooled populations and the protein interactome of individual glycoRNA species remain unresolved. Here we develop GlycoRNA-Map, a programmable photocatalytic proximity-labeling method that maps proteins surrounding sequence-defined endogenous glycoRNAs on living cells. A sialic-acid-binding aptamer and an RNA-hybridization probe jointly recruit a fluorophore-modified oligonucleotide through dual recognition of the glycan and RNA moieties of glycoRNAs. Light-activated biotinylation followed by quantitative mass spectrometry identified 200-418 enriched proteins for each of five glycoRNAs in A549 cells. Comparison of these proteomes revealed limited overlap among different glycoRNA sequences and across cell types, demonstrating that glycoRNA-associated protein environments depend on both RNA identity and cellular context. Transforming growth factor-{beta} receptors 1 and 2 (TGFBR1 and TGFBR2) were identified in the proximal proteomes of multiple glycoRNAs. Removal of cell-surface RNAs enhanced TGF-{beta} receptor signaling and promoted cell migration, demonstrating that glycoRNAs can functionally regulate extracellular signaling by interacting with their protein neighborhoods. GlycoRNA-Map thus provides an accessible strategy for resolving sequence-specific glycoRNA-protein interactome and discovering their roles in membrane signaling.

Newcastle disease virus (NDV) continues to cause major economic losses in poultry, driving many recent virulent outbreaks. Maternally derived antibodies (MDA) can interfere with the development of active immunity following vaccination in young chicks. NDV vaccine strains that are safe for day-old chicks are not safe for administration to embryos. Here, we combined a computationally guided attenuation strategy with an in-ovo vaccination and challenge study to develop and evaluate an embryo-safe NDV vaccine candidate. Using a large NDV sequence compendium, we applied a local RNA folding energy based algorithm to identify folding selected clusters within the NDV fusion protein; we introduced targeted synonymous substitutions within the first ~900 nucleotides to modulate local RNA structure while preserving the encoded protein sequence. The recombinant attenuated variant was propagated in embryonated chicken eggs to generate vaccine stocks for in-ovo vaccination. After 18 days of embryonation, the synthetic variants were well tolerated, achieving more than 97% hatchability, with no pre-challenge morbidity or mortality, while unvaccinated challenge controls showed complete mortality. Together, these data support a model in which structure-aware synonymous modifications can yield an embryo-safe NDV backbone suitable for in-ovo delivery, with protective efficacy exhibiting a clear dose window under high MDA conditions.

During phage infection, phages reprogram their host to secure energy, building blocks for their genome, and proteins, as well as the transcription and translation machinery required to replicate their genome and produce phage particles. This also includes reprogramming the host transcriptome to modify host gene expression and align it with the needs of the phage during infection. In this study, we present a putative early-expressed protein, Gp204 of Mosigvirus AV110, renamed Host transcription modulator (Htm), that has a broad impact on the extended-spectrum beta-lactamase (ESBL)- associated E. coli transcriptome. Overexpression of Htm decreased the growth rate of ESBL-E. coli and caused DNA condensation. Htm is structurally similar to bacterial enhancer-binding proteins, binds ssDNA, and alters the expression levels of over 700 E. coli genes when expressed. The majority of these genes were upregulated and are involved in multiple pathways, such as galactose metabolism and pentose and glucuronate interconversions, which may promote the synthesis of purines, pyrimidines, and histidine, all of which are required for the replication, transcription, and translation of the phage genome. Furthermore, energy production is likely to be upregulated by increased availability of acetyl-CoA, resulting from, among other factors, fatty acid degradation. Htm also downregulated the expression of the structural genes involved in flagellar assembly and multiple two-component systems. In conclusion, Htm could promote phage progeny production by increasing the availability of nucleotides, amino acids, and energy, while inhibiting non-essential processes, such as flagella formation.

Glucoamylase is essential for industrial starch saccharification, but the limited thermostability and near-neutral pH tolerance of fungal glucoamylases necessitate cooling and acidification of liquefied starch. Here, we developed an artificial intelligence-guided strategy to simultaneously improve the thermostability, pH tolerance, and catalytic activity of glucoamylase from Penicillium oxalicum (PoGA). Two property-specific machine-learning models, CASPE-T and CASPE-A, identified substitutions associated with thermostability and pH tolerance, respectively. Experimental screening identified beneficial substitutions in 11 of 21 CASPE-T and 12 of 22 CASPE-A candidates. Folding-energy-guided recombination integrated the two traits while maintaining structural compatibility. The optimal variant, PoGA T513E/Q305N, exhibited 2.21-fold higher specific activity than the wild type, with half-life extended from 22.3 to 57.9 min at 60 degrees C and from 16.6 to 64.7 min at pH 8.0. Molecular dynamics simulations attributed these improvements to reinforcement of high-occupancy hydrogen-bonding networks, suppression of conformational fluctuations in the linker and carbohydrate-binding module, enhanced long-range dynamic coordination, and preservation of a compact catalytic architecture. At 60 degrees C and pH 6.5 without acidification, PoGA T513E/Q305N produced 219.9 g/L glucose and achieved 89.1% starch conversion, 31.4% higher than the wild type. This work provides an efficient framework for multi-objective enzyme engineering and sustainable starch biorefining.

The 5'-untranslated region (5'-UTR) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly structured and comprises five conserved stem-loops that serve as cis-acting regulatory elements. During infection, host proteins are recruited to these elements and regulate multiple steps during the viral life cycle. Stem-loop 5 (SL5) is highly conserved among different alpha- and betacoronaviruses and includes the start codon of non-structural protein 1 (nsp1). However, the function of SL5 in viral replication and its interacting proteins remain to be determined. Here, we identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a host protein functionally interacting with SL5. HNRNPM translocates into the cytoplasm during SARS-CoV-2 infection and promotes protein synthesis from the viral 5'-UTR. Mutations in the lower stem of SL5 impair HNRNPM binding and reduce protein synthesis. Notably, the interaction between HNRNPM and SL5 is conserved among different alpha- and betacoronaviruses. Our findings indicate that HNRNPM is an important host factor that regulates viral translation to promote SARS-CoV-2 replication.

Components of nuclear pore complexes are broadly classified into two categories: intrinsically disordered FG-repeat nucleoporins such as Nup98, which can undergo liquid-liquid phase separation, and scaffold nucleoporins, which form the NPC's structural framework. The basis for crosstalk of these nucleoporins within NPC and beyond is poorly understood. Here we show that human Nup188, a helical inner-ring scaffold protein, forms condensates both in purified form as well as at endogenous level. This condensate exhibits fusion, dissolved in 1,6-hexanediol and recovered after photobleaching, with dynamics and material properties comparable to those of hNup98 condensates. Moreover, these condensates are not colocalized to annulate lamellae. Our analysis also revealed that hNup188CTD is sufficient to form condensates and MD simulations revealed the key roles of IDR1-3 for condensation. Notably, hNup93 expression along with hNup188 suppressed the condensate formation. Together, these findings indicate that a structured scaffold nucleoporin can adopt a condensate-forming state via its CTD and Nup93 binding can suppress this phenomenon.

Vibrio coralliilyticus (V. coralliilyticus) is an emerging coral pathogen associated with tissue-loss diseases in reef-building corals and diseases in other marine organisms, such as oyster larvae. Despite the great ecological and economic significance of this pathogenic bacterium, characterization of bacteriophages infecting V. coralliilyticus remains limited, particularly of temperate phages. In this study, we describe the isolation and characterization of four novel phages - Vibrio phages Satoshi, Kolob, Ushijima, and Kane - obtained from coastal waters near L[a]ie, Hawaii, using the indigenous Hawaiian V. coralliilyticus strain OCN008 as the host. All four phages produced small, turbid plaques and encoded integrases, consistent with temperate lifestyles. The genome organization and taxonomic relationships of the viruses were examined using whole-genome sequencing and comparative genomics, while representative virion morphologies were examined by transmission electron microscopy. To the best of the authors' knowledge, these are the first V. coralliilyticus phages reported from Oahu, Hawaii. Comparative genomic analyses support the placement of all four phages within the class Caudoviricetes, but outside of any previously described orders, families, or genera within the class. Satoshi, Kolob, and Ushijima, along with previously described V. coralliilyticus phage CKB-S2, cluster within one genus-level lineage, while Kane, which has no close relatives in available databases, represents a distinct, previously undescribed lineage. All four phages were also found to contain homologs to experimentally established virulence factors, which may contribute to the pathogenicity of the host bacterium during lysogeny. Together, these isolates expand the diversity of phages infecting V. coralliilyticus and support the proposal of two new genera of Caudoviricetes: the genus Mokushimavirus, containing Satoshi, Kolob, Ushijima, and CKB-S2, and the genus Laievirus, containing Kane.

T cell receptor (TCR) specificity prediction is critical for understanding adaptive immunity and for enabling the development of therapeutic interventions. Current machine learning models for predicting TCR-peptide-MHC interactions perform poorly, despite large efforts by many groups and advances in protein language models. This work identifies and addresses the fundamental issues that might be hindering progress in this field, focusing on current approaches for constructing training and validation sets, particularly on the strategies used by commonly used methods, which generate negative data either by shuffling TCRs between epitopes or by pairing epitopes with TCRs from background datasets of healthy donors. Here, we visualize and quantify the overlap between positive and generated negative TCR sets produced by both strategies using a range of sequence embedding approaches, including simple encodings (one-hot and BLOSUM62), general protein language models (ESM-2 and ProtGPT2), and a TCR-specific embedding model (TCR-BERT). Using paired {beta}TCR data from VDJdb for two well-studied epitopes (GILGFVFTL and KLGGALQAK), we train Random Forest classifiers on each embedding representation and evaluate their ability to distinguish positive from negative data examples. We find that general sequence embedding methods struggle to differentiate between positive and negative TCRs. We further show that the choice of negative data generation method significantly impacts classifier performance. Our findings highlight fundamental limitations in current TCR specificity training data and general protein language models, and underscore the need for TCR-specific embeddings, experimentally validated negative datasets, transparent reporting of negative data construction, and epitope-specific modeling approaches.

Human coronaviruses (HCoVs) cause pathogenic outcomes ranging from mild illness to severe respiratory disease. Determining how respiratory cells coordinate an early antiviral response is necessary to understand successful control of HCoV replication. The Type I Interferon (IFN) response is a major component of innate viral immunity. IFN signaling leads to the upregulation of hundreds of interferon-stimulated genes (ISGs) that can have antiviral activity, though the individual ISGs that are responsible for restricting mildly pathogenic HCoVs in the respiratory epithelium are not clear. Here, we developed a targeted CRISPR-Cas9 knockout sgRNA library (termed the respISG library), focusing on ISGs that are upregulated across a panel of respiratory cells, to screen for innate host factors that restrict the mildly pathogenic human coronavirus, HCoV-OC43. We executed cell death-based screens in immortalized human small airway epithelial cells and identified lymphocyte antigen 6E (LY6E) as a top hit. We confirmed LY6E activity in validation studies and found it regulates entry of various SARS-CoV-2 Spike-pseudotyped lentiviruses. In addition to demonstrating a key role for LY6E in blocking a variety of HCoVs in small airway epithelial cells, this study establishes a loss-of-function CRISPR-Cas9 screening platform for identification of HCoV antiviral ISGs in respiratory cells.